Inbred corn line ML12

ABSTRACT

An inbred corn line, designated ML12, is disclosed. The invention relates to the seeds of inbred corn line ML12, to the plants and plant parts of inbred corn line ML12 and to methods for producing a corn plant, either inbred or hybrid, by crossing inbred corn line ML12 with itself or another corn line. The invention also relates to products produced from the seeds, plants, or parts thereof, of inbred corn line ML12 and/or of the hybrids produced using the inbred as a parent. The invention further relates to methods for producing a corn plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other inbred corn lines derived from inbred corn line ML12.

FIELD OF THE INVENTION

The present invention relates to a new and distinctive corn inbred line(Zea mays, also known as maize), designated ‘ML12’.

BACKGROUND OF THE INVENTION

The disclosures, including the claims, figures and/or drawings, of eachand every patent, patent application, and publication cited herein arehereby incorporated herein by reference in their entireties.

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety or hybridan improved combination of desirable traits from the parental germplasm.These important traits may include higher yield, resistance to diseasesand insects, better stalks and roots, tolerance to drought and heat,reduction of grain moisture at harvest as well as better agronomicquality. With mechanical harvesting of many crops, uniformity of plantcharacteristics such as germination and stand establishment, growthrate, maturity and plant and ear height is important.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection, andbackcross breeding.

The complexity of inheritance influences choice of breeding method.Backcross breeding is used to transfer one or a few favorable genes fora heritable trait into a desirable cultivar. This approach has been usedextensively for breeding disease-resistant cultivars; nevertheless, itis also suitable for the adjustment and selection of morphologicalcharacters, color characteristics and simply inherited quantitativecharacters such as earliness, plant height or seed size and shape.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested per se and inhybrid combination and compared to appropriate standards in environmentsrepresentative of the commercial target area(s) for three or more years.The best lines are candidates for use as parents in new commercialcultivars; those still deficient in a few traits may be used as parentsto produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to twelve years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a focus on clear objectives.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of corn breeding is to develop new, unique and superior corninbred lines and hybrids. The breeder initially selects and crosses twoor more parental lines, followed by repeated self pollination or selfingand selection, producing many new genetic combinations. Another methodused to develop new, unique and superior corn inbred lines and hybridsoccurs when the breeder selects and crosses two or more parental lines,followed by haploid induction and chromosome doubling that results inthe development of dihaploid inbred lines. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations and the same is true for the utilization of thedihaploid breeding method.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The inbred lineswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures or dihaploidbreeding procedures), and with millions of different possible geneticcombinations being generated. A breeder of ordinary skill in the artcannot predict the final resulting lines he develops, except possibly ina very gross and general fashion. This unpredictability results in theexpenditure of large research funds to develop a superior new corninbred line.

The development of commercial corn hybrids requires the development ofhomozygous inbred lines, the crossing of these lines, and the evaluationof the crosses. Pedigree breeding and recurrent selection breedingmethods are used to develop inbred lines from breeding populations.Breeding programs combine desirable traits from two or more inbred linesor various broad-based sources into breeding pools from which inbredlines are developed by selfing and selection of desired phenotypes orthrough the dihaploid breeding method followed by the selection ofdesired phenotypes. The new inbreds are crossed with other inbred linesand the hybrids from these crosses are evaluated to determine which havecommercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars. Similarly, the development of new inbred linesthrough the dihaploid system requires the selection of the best inbredsfollowed by four to five years of testing in hybrid combinations inreplicated plots.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable cultivar or inbredline which is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., R. W. Allard, 1960, Principles of Plant Breeding, JohnWiley and Son; Briggs, F. N. and Knowles, P. F. 1967. Introduction toPlant Breeding. Reinhold Publishing Corporation; N. W. Simmonds, 1979,Principles of Crop Improvement, Longman Group Limited; W. R. Fehr, 1987,Principles of Crop Development, Macmillan Publishing Co.; N. F. Jensen,1988, Plant Breeding Methodology, John Wiley & Sons).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F₁progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F₁ hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor exhibited by F₁ hybrids islost in the next generation (F₂). Consequently, seed from hybridvarieties is not used for planting stock.

Hybrid corn seed is typically produced by a male sterility system or byincorporating manual or mechanical detasseling. Alternate strips of twocorn inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Providing that there issufficient isolation from sources of foreign corn pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

The laborious, and occasionally unreliable, detasseling process can beavoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMSinbred are male sterile as a result of factors resulting from thecytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent incorn plants, since only the female provides cytoplasm to the fertilizedseed. CMS plants are fertilized with pollen from another inbred that isnot male-sterile. Pollen from the second inbred may or may notcontribute genes that make the hybrid plants male-fertile. Seed fromdetasseled fertile corn and CMS produced seed of the same hybrid can beblended to insure that adequate pollen loads are available forfertilization when the hybrid plants are grown.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. These and all patents referred toare incorporated by reference. In addition to these methods, Albertsenet al., U.S. Pat. No. 5,432,068, have developed a system of nuclear malesterility which includes: identifying a gene which is critical to malefertility, silencing this native gene which is critical to malefertility; removing the native promoter from the essential malefertility gene and replacing it with an inducible promoter; insertingthis genetically engineered gene back into the plant; and thus creatinga plant that is male sterile because the inducible promoter is not “on”resulting in the male fertility gene not being transcribed. Fertility isrestored by inducing, or turning “on,” the promoter, which in turnallows the gene that confers male fertility to be transcribed.

There are many other methods of conferring genetic male sterility in theart, each with its own benefits and drawbacks. These methods use avariety of approaches such as delivering into the plant a gene encodinga cytotoxic substance associated with a male tissue specific promoter oran anti-sense system in which a gene critical to fertility is identifiedand an antisense to that gene is inserted in the plant (see,Fabinjanski, et al. EPO 89/0301053.8 publication number 329,308 and PCTapplication PCT/CA90/00037 published as WO 90/08828).

Another version useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see Carlson, G. R., U.S. Pat. No. 4,936,904). Application ofthe gametocide, timing of the application, and genotype often limit theusefulness of the approach.

Corn is an important and valuable field crop. Thus, a continuing goal ofplant breeders is to develop stable, high yielding corn hybrids that areagronomically sound. The reasons for this goal are obviously to maximizethe amount of ears or kernels produced on the land used and to supplyfood for both humans and animals. To accomplish this goal, the cornbreeder must select and develop corn plants that have the traits thatresult in superior parental lines for producing hybrids.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary and illustrative, not limiting in scope. In variousembodiments, one or more of the above-described problems have beenreduced or eliminated, while other embodiments are directed to otherimprovements.

According to the invention, there is provided an inbred corn linedesignated ML12. This invention thus relates to the seeds of inbred cornline ML12, to the plants or parts thereof of inbred corn line ML12, toplants or parts thereof having all the physiological and morphologicalcharacteristics of inbred corn line ML12 and to plants or parts thereofhaving all the physiological and morphological characteristics of inbredcorn line ML12 listed in Table 1, including but not limited to asdetermined at the 5% significance level when grown in the sameenvironmental conditions. The invention also relates to variants,mutants and trivial modifications of the seed or plant of inbred cornline ML12. Parts of the inbred corn plant of the present invention arealso provided, such as e.g., pollen obtained from an inbred plant and anovule of the inbred plant.

The plants and seeds of the present invention include those that may beof an essentially derived variety as defined in section 41(3) of thePlant Variety Protection Act, i.e., a variety that:

-   -   (i) is predominantly derived from inbred corn line ML12 or from        a variety that is predominantly derived from inbred corn line        ML12, while retaining the expression of the essential        characteristics that result from the genotype or combination of        genotypes of inbred corn line ML12;    -   (ii) is clearly distinguishable from inbred corn line ML12; and    -   (iii) except for differences that result from the act of        derivation, conforms to the initial variety in the expression of        the essential characteristics that result from the genotype or        combination of genotypes of the initial variety.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of inbred corn plant ML12. The tissue culture willpreferably be capable of regenerating plants having all thephysiological and morphological characteristics of the foregoing inbredcorn plant. Preferably, the cells of such tissue cultures will beembryos, ovules, meristematic cells, seeds, callus, pollen, leaves,anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks,stalks or the like. Protoplasts produced from such tissue culture arealso included in the present invention. The corn shoots, roots and wholeplants regenerated from the tissue cultures are also part of theinvention.

Also included in this invention are methods for producing a corn plantproduced by crossing the inbred corn line ML12 with itself or anothercorn line. When crossed with itself, i.e., crossed with another inbredcorn line ML12 plant or self-pollinated, the inbred line ML12 will beconserved (e.g., as an inbred). When crossed with another, differentcorn line, an F₁ hybrid seed is produced. F₁ hybrid seeds and plantsproduced by growing said hybrid seeds are included in the presentinvention. A method for producing an F₁ hybrid corn seed comprisingcrossing inbred corn line ML12 corn plant with a different corn plantand harvesting the resultant hybrid corn seed are also part of theinvention. The hybrid corn seed produced by the method comprisingcrossing inbred corn line ML12 corn plant with a different corn plantand harvesting the resultant hybrid corn seed are included in theinvention, as are included the hybrid corn plant or parts thereof, seedsincluded, produced by growing said hybrid corn seed.

In another embodiment, this invention relates to a method for producingthe inbred corn line ML12 from a collection of seeds, the collectioncontaining both inbred corn line ML12 seeds and hybrid seeds havinginbred corn line ML12 as a parental line. Such a collection of seedmight be a commercial bag of seeds. Said method comprises planting thecollection of seeds. When planted, the collection of seeds will produceinbred corn line ML12 plants from inbred corn line ML12 seeds and hybridplants from hybrid seeds. The plants having all the physiological andmorphological characteristics of corn inbred corn line ML12 or having adecreased vigor compared to the other plants grown from the collectionof seeds are identified as inbred corn line ML12 parent plants. Saiddecreased vigor is due to the inbreeding depression effect and can beidentified for example by a less vigorous appearance for vegetativeand/or reproductive characteristics including shorter plant height,small ear size, ear and kernel shape, ear color or othercharacteristics. As previously mentioned, if the inbred corn line ML12is self-pollinated, the inbred corn line ML12 will be preserved,therefore, the next step is controlling pollination of the inbred parentplants in a manner which preserves the homozygosity of said inbred cornline ML12 parent plant and the final step is to harvest the resultantseed.

This invention also relates to methods for producing other inbred cornlines derived from inbred corn line ML12 and to the inbred corn linesderived by the use of those methods.

In another aspect, the present invention provides transformed inbredcorn line ML12 or parts thereof that have been transformed so that itsgenetic material contains one or more transgenes, preferably operablylinked to one or more regulatory elements. Also, the invention providesmethods for producing a corn plant containing in its genetic materialone or more transgenes, preferably operably linked to one or moreregulatory elements, by crossing transformed inbred corn line ML12 witheither a second plant of another corn line, or a non-transformed cornplant of the inbred corn line ML12, so that the genetic material of theprogeny that results from the cross contains the transgene(s),preferably operably linked to one or more regulatory elements. Theinvention also provides methods for producing a corn plant that containsin its genetic material one or more transgene(s), wherein the methodcomprises crossing the inbred corn line ML12 with a second plant ofanother corn line which contains one or more transgene(s) operablylinked to one or more regulatory element(s) so that the genetic materialof the progeny that results from the cross contains the transgene(s)operably linked to one or more regulatory element(s). Transgenic cornplants, or parts thereof produced by the method are in the scope of thepresent invention.

More specifically, the invention comprises methods for producing cornplants or seeds with at least one trait selected from the groupconsisting of male sterile, male fertile, herbicide resistant, insectresistant, disease resistant, water stress tolerant corn plants orseeds, or corn plants or seeds with modified, in particular decreased,phytate content, with modified waxy and/or amylose starch content, withmodified protein content, with modified oil content or profile, withincreased digestibility or with increased nutritional quality. Saidmethods comprise transforming the inbred corn line ML12 corn plant withnucleic acid molecules that confer, for example, male sterility, malefertility, herbicide resistance, insect resistance, disease resistance,water stress tolerance, or that can modify the phytate, the waxy and/oramylose starches, the protein or the oil contents, the digestibility orthe nutritional qualities, respectively. The transformed corn plants orseeds obtained from the provided methods, including, for example, thosecorn plants or seeds with male sterility, male fertility, herbicideresistance, insect resistance, disease resistance, water stresstolerance, modified phytate, waxy and/or amylose starches, protein oroil contents, increased digestibility and increased nutritional qualityare included in the present invention. Plants may display one or more ofthe above listed traits. For the present invention and the skilledartisan, disease is understood to be fungal disease, viral disease,bacterial disease or other plant pathogenic diseases and diseaseresistant plant encompasses plants resistant to fungal, viral, bacterialand other plant pathogens.

Also included in the invention are methods for producing a corn plant orseed containing in its genetic material one or more transgenes involvedwith fatty acid metabolism, carbohydrate metabolism, and starch contentsuch as waxy starch or increased amylose starch. The transgenic cornplants or seeds produced by these methods are also part of theinvention.

In another aspect, the present invention provides for methods ofintroducing one or more desired trait(s) into the inbred corn line ML12and plants or seeds obtained from such methods. The desired trait(s) maybe, but not exclusively, a single gene, preferably a dominant but also arecessive allele. Preferably, the transferred gene or genes will confersuch traits as male sterility, herbicide resistance, insect resistance,resistance for bacterial, fungal, or viral disease, male fertility,water stress tolerance, enhanced nutritional quality, modified waxycontent, modified amylose content, modified protein content, modifiedoil content, enhanced plant quality, enhanced digestibility andindustrial usage. The gene or genes may be naturally occurring maizegene(s) or transgene(s) introduced through genetic engineeringtechniques. The method for introducing the desired trait(s) ispreferably a backcrossing process making use of a series of backcrossesto the inbred corn line ML12 during which the desired trait(s) ismaintained by selection.

When using a transgene, the trait is generally not incorporated intoeach newly developed line such as inbred corn line ML12 by directtransformation. Rather, the more typical method used by breeders ofordinary skill in the art to incorporate the transgene is to take a linealready carrying the transgene and to use such line as a donor line totransfer the transgene into the newly developed line. The same wouldapply for a naturally occurring trait (e.g., a native trait, such as butnot limited to drought tolerance or improved nitrogen utilization) orone arising from spontaneous or induced mutations. The backcrossbreeding process comprises the following steps: (a) crossing inbred cornline ML12 plants with plants of another line that comprise the desiredtrait(s), (b) selecting the F₁ progeny plants that have the desiredtrait(s); (c) crossing the selected F₁ progeny plants with the inbredcorn line ML12 plants to produce backcross progeny plants; (d) selectingfor backcross progeny plants that have the desired trait(s) andphysiological and morphological characteristics of corn inbred corn lineML12 to produce selected backcross progeny plants; and (e) repeatingsteps (c) and (d) one, two, three, four, five, six, seven, eight, nineor more times in succession to produce selected, second, third, fourth,fifth, sixth, seventh, eighth, ninth or higher backcross progeny plantsthat comprise the desired trait(s) and all the physiological andmorphological characteristics of corn inbred line ML12 as listed inTable 1, including but not limited to at a 5% significance level whengrown in the same environmental conditions. The corn plants or seedsproduced by the methods are also part of the invention. Backcrossingbreeding methods, well known to one skilled in the art of plant breedingwill be further developed in subsequent parts of the specification.

In another aspect of the invention, inbred corn line ML12 may be used asa parent, or a single gene conversion or a transgenic inbred corn lineof ML12, such as ML12 MON89034, may be used as a parent and may becrossed with another corn line including but not limited to thefollowing corn lines: AB10, BB36, BC110, and BC115. Corn inbred ML12 maybe used as a parent or a single gene conversion or a transgenic inbredline of ML12 such as ML12 MON89034 may be used as a parent and may becrossed with another corn line including but not limited to thefollowing corn lines: single gene conversions or transgenic inbred linesof BB36, such as BB36 MON603, single gene conversions or transgenicinbred lines of BC110, such as BC110 MON88017, single gene conversionsor transgenic inbred lines of BC115, such as BC115 MON603, or transgenicinbred lines of AB 10, such as AB10 MON603. Preferably, the single geneconversions or transgenic inbred lines will confer such traits,herbicide resistance, insect resistance, resistance for bacterial,fungal, or viral disease, male fertility, water stress tolerance,enhanced nutritional quality, modified waxy content, modified amylosecontent, modified protein content, modified oil content, enhanced plantquality, enhanced digestibility and industrial usage. The gene or genesmay be naturally occurring maize gene(s) (e.g., native traits) ortransgene(s) introduced through genetic engineering techniques. Thehybrid corn plants or seeds having inbred corn line ML12 or a singlegene conversion or a transgenic inbred corn line ML12 as a parental lineand having another, different, corn line as a second parental line asdiscussed above are comprised in the present invention.

Any DNA sequence(s), whether from a different species or from the samespecies that is inserted into the genome using transformation isreferred to herein collectively as “transgenes.” In some embodiments ofthe invention, a transformed variant of ML12 may contain at least onetransgene but could contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10transgenes. In another embodiment of the invention, a transformedvariant of another corn line used as the other parental line may containat least one transgene but could contain at least 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 transgenes, such as LH287, MON810, and MON89034.

In an embodiment of this invention is a method of making a backcrossconversion of inbred corn line ML12, comprising the steps of crossing aplant of corn inbred corn line ML12 with a donor plant comprising amutant gene or transgene conferring a desired trait, selecting an F₁progeny plant comprising the mutant gene or transgene conferring thedesired trait, and backcrossing the selected F₁ progeny plant to a plantdf inbred corn line ML12. This method may further comprise the step ofobtaining a molecular marker profile of inbred corn line ML12 and usingthe molecular marker profile to select for a progeny plant with thedesired trait and the molecular marker profile of inbred corn line ML12.In the same manner, this method may be used to produce an F₁ hybrid seedby adding a final step of crossing the desired trait conversion ofinbred corn line ML12 with a different corn plant to make F₁ hybrid cornseed comprising a mutant gene or transgene conferring the desired trait.

In some embodiments of the invention, the number of loci that may bebackcrossed into inbred corn line ML12 is at least 1, 2, 3, 4, or 5. Asingle locus may contain several transgenes, such as a transgene fordisease resistance that, in the same expression vector, also contains atransgene for herbicide resistance. The gene for herbicide resistancemay be used as a selectable marker and/or as a phenotypic trait. Asingle locus conversion of site specific integration system allows forthe integration of multiple genes at the converted locus.

In a preferred embodiment, the present invention provides methods forincreasing and producing inbred corn line ML12 seed, whether by crossinga first inbred parent corn plant with a second inbred parent corn plantand harvesting the resultant corn seed, wherein both said first andsecond inbred corn plant are the inbred corn line ML12 or by planting aninbred corn seed of the inbred corn line ML12, growing an inbred cornline ML12 plant from said seed, controlling a self pollination of theplant where the pollen produced by the grown inbred corn line ML12 plantpollinates the ovules produced by the very same inbred corn line ML12grown plant and harvesting the resultant seed.

The invention further provides methods for developing corn plants in acorn plant breeding program using plant breeding techniques includingrecurrent selection, backcrossing, pedigree breeding, molecular marker(Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms(RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs). AmplifiedFragment Length Polymorphisms (AFLPs), and Simple Sequence Repeats(SSRs) which are also referred to as Microsatellites, etc.) enhancedselection, genetic marker enhanced selection and transformation. Cornseeds, plants, and parts thereof produced by such breeding methods arealso part of the invention.

In addition, any and all products made using the corn seeds, plants andparts thereof obtained from inbred corn line ML12 or from any corn lineproduced using inbred corn line ML12 as a direct or indirect parent arealso part of the invention. Examples of such corn products include butare not limited to corn meal, corn flour, corn starch, corn syrup, cornsweetener and corn oil. The origin of the corn used in such cornproducts can be determined by tracking the source of the corn used tomake the products and/or by using protein (isozyme, ELISA, etc.) and/orDNA (RFLP, PCR, SSR, SNP, EST, etc.) testing.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. The allele is any of one or more alternative forms of a gene,all of which relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Collection of seeds. In the context of the present invention acollection of seeds will be a grouping of seeds mainly containingsimilar kind of seeds, for example hybrid seeds having the inbred lineof the invention as a parental line, but that may also contain, mixedtogether with this first kind of seeds, a second, different kind ofseeds, of one of the inbred parent lines, for example the inbred line ofthe present invention. A commercial bag of hybrid seeds having theinbred line of the invention as a parental line and containing also theinbred line seeds of the invention would be, for example such acollection of seeds.

Daily heat unit value. The daily heat unit value (also referred to asgrowing degree unit, or GDU) is calculated as follows: (the maximumdaily temperature+the minimum daily temperature)/2 minus 50. Alltemperatures are in degrees Fahrenheit. The maximum temperaturethreshold is 86 degrees, if temperatures exceed this, 86 is used. Theminimum temperature threshold is 50 degrees, if temperatures go belowthis, 50 is used. For each hybrid, it takes a certain number of GDUs toreach various stages of plant development. GDUs are a way of measuringplant maturity. GDUs can also relate to stages of growth for an inbredline.

Decreased vigor. A plant having a decreased vigor in the presentinvention is a plant that, compared to other plants has a less vigorousappearance for vegetative and/or reproductive characteristics includingshorter plant height, small ear size, ear and kernel shape, ear color orother characteristics.

Dropped ears. This is a measure of the number of dropped ears per plot,and represents the percentage of plants that dropped an ear prior toharvest.

Dry down. This is the rate at which a hybrid will reach acceptableharvest moisture.

Ear height. The ear height is a measure from the ground to the upper earnode attachment, and is measured in centimeters.

Essentially all of the physiological and morphological characteristics.A plant having essentially all of the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

GDU pollen. The number of heat units from planting until 50% of theplants in the hybrid are shedding pollen.

GDU silk. The GDU silk (=heat unit silk) is the number of growing degreeunits (GDU) or heat units required for an inbred line or hybrid to reachsilk emergence from the time of planting.

Harvest aspect. This is a visual rating given the day of harvest or theprevious day. Hybrids are rated 1 (poorest) to 9 (best) with poorerscores given for poor plant health, visible signs of fungal infection,poor plant intactness characterized by missing leaves, tassels, or othervegetative parts, or a combination of these traits.

Herbicide resistant or tolerant. A plant containing anyherbicide-resistant gene or any DNA molecule or construct (or replicatethereof) which is not naturally occurring in the plant which results inincrease tolerance to any herbicide including, imidazoline,sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine andbenzonitrile. For purposes of this definition, a DNA molecule orconstruct shall be considered to be naturally occurring if it exists ina plant at a high enough frequency to provide herbicide resistancewithout further selection and/or if it has not been produced as a resultof tissue culture selection, mutagenesis, genetic engineering usingrecombinant DNA techniques or other in vitro or in vivo modification tothe plant.

Inbreeding depression. The inbreeding depression is the loss ofperformance of the inbreds due to the effect of inbreeding, i.e., due tothe mating of relatives or to self-pollination. It increases thehomozygous recessive alleles leading to plants which are weaker andsmaller and having other less desirable traits.

Late plant greenness. Similar to a stay green rating. This is a visualassessment given at around the dent stage but typically a few weeksbefore harvest to characterize the degree of greenness left in theleaves. Plants are rated from 1 (poorest) to 9 (best) with poorer scoresgiven for plants that have more non-green leaf tissue typically due toearly senescence or from disease.

MN RM. This represents the Minnesota Relative Maturity Rating (MN RM)for the hybrid and is based on the harvest moisture of the grainrelative to a standard set of checks of previously determined MN RMrating. Regression analysis is used to compute this rating.

Moisture. The moisture is the actual percentage moisture of the grain atharvest.

Plant cell. Plant cell, as used herein includes plant cells whetherisolated, in tissue culture, or incorporated in a plant or plant part.

Plant habit. This is a visual assessment assigned during the latevegetative to early reproductive stages to characterize the plant's leafhabit. It ranges from decumbent with leaves growing horizontally fromthe stalk to a very upright leaf habit, with leaves growing nearvertically from the stalk.

Plant height. This is a measure of the height of the hybrid from theground to the tip of the tassel, and is measured in centimeters.

Plant intactness. This is a visual assessment assigned to a hybrid orinbred at or close to harvest to indicate the degree that the plant hassuffered disintegration through the growing season. Plants are ratedfrom 1 (poorest) to 9 (best) with poorer scores given for plants thathave more of their leaf blades missing.

Plant part. As used herein, the term “plant part” includes any part ofthe plant including but not limited to leaves, stems, roots, seeds,grains, embryos, pollens, ovules, flowers, ears, cobs, husks, stalks,root tips, anthers, silk, tissue, cells and the like.

Pollen shed. This is a visual rating assigned at flowering to describethe abundance of pollen produced by the anthers. Inbreds are rated 1(poorest) to 9 (best) with the best scores for inbreds with tassels thatshed more pollen during anthesis.

Post-anthesis root lodging. This is a percentage plants that root lodgeafter anthesis: plants that lean from the vertical axis at anapproximately 30° angle or greater.

Pre-anthesis brittle snapping. This is a percentage of “snapped” plantsfollowing severe winds prior to anthesis.

Pre-anthesis root lodging. This is a percentage plants that root lodgeprior to anthesis: plants that lean from the vertical axis at anapproximately 30° angle or greater.

Predicted RM. This trait for a hybrid, predicted relative maturity (RM),is based on the harvest moisture of the grain. The relative maturityrating is based on a known set of checks and utilizes conventionalmaturity such as the Comparative Relative Maturity Rating System or itssimilar, the Minnesota Relative Maturity Rating System.

Quantitative trait loci (QTL). Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Root lodging. The root lodging is the percentage of plants that rootlodge; i.e., those that lean from the vertical axis at an approximate30° angle or greater would be counted as root lodged.

Seed quality. This is a visual rating assigned to the kernels of theinbred. Kernels are rated 1 (poorest) to 9 (best) with poorer scoresgiven for kernels that are very soft and shriveled with splitting of thepericarp visible and better scores for fully formed kernels.

Seedling vigor. This is the vegetative growth after emergence at theseedling stage, approximately five leaves.

Silking ability. This is a visual assessment given during flowering.Plants are rated on the amount and timing of silk production. Plants arerated from 1 (poorest) to 9 (best) with poorer scores given for plantsthat produce very little silks that are delayed past pollen shed.

Single gene converted. Single gene converted or conversion plants refersto plants which are developed by a plant breeding technique calledbackcrossing wherein essentially all the morphological and physiologicalcharacteristics of an inbred are recovered in addition to the singlegene transferred into the inbred via the backcrossing technique or viagenetic engineering. This also includes multiple transference of singlegenes.

Stalk lodging. This is the percentage of plants that stalk lodge, i.e.,stalk breakage, as measured by either natural lodging or pushing thestalks and determining the percentage of plants that break off below theear. This is a relative rating of a hybrid to other hybrids forstandability.

Standability. A term referring to the how well a plant remains uprighttowards the end of the growing season. Plants with excessive stalkbreakage and/or root lodging would be considered to have poorstandability.

Stay green. Stay green is the measure of plant health near the time ofblack layer formation (physiological maturity). A high score indicatesbetter late-season plant health.

Transgenic. Where an inbred line has been converted to contain one ormore transgenes by single gene conversion or by direct transformation.

Variety. A plant variety as used by one skilled in the art of plantbreeding means a plant grouping within a single botanical taxon of thelowest known rank which can be defined by the expression of thecharacteristics resulting from a given genotype or combination ofphenotypes, distinguished from any other plant grouping by theexpression of at least one of the said characteristics and considered asa unit with regard to its suitability for being propagated unchanged(International Convention for the Protection of New Varieties ofPlants).

Yield (Bushels/Acre). The yield is the actual yield of the grain atharvest adjusted to 15.5% moisture.

DETAILED DESCRIPTION OF THE INVENTION

Inbred corn line ML12 is a yellow dent corn inbred with superiorcharacteristics, and provides a very good male parental line in crossesfor producing first generation (F₁) hybrid corn.

Inbred corn line ML12 is best adapted to the East, Central, South andWestern regions of the United States Corn Belt in the zones that arecommonly referred to as Zones 5, 6 and 7. Hybrids that are adapted tothese maturity zones can be grown on a significant number of acres as itrelates to the total of the USA corn acres. Inbred corn line ML12 can beused to produce hybrids having a relative maturity of approximately 100to 110 days on the Minnesota Relative Maturity Rating System for harvestmoisture of grain. Inbred corn line ML12 produces an above averagevolume of pollen as a male parent in the production of F₁ hybrids.Inbred corn line ML12 shows good emergence vigor, and F1 hybridsfathered by ML12 dry down extremely rapidly compared to other hybridswith similar GDU silk data. Thus inbred corn line ML12, when compared toMo17/LH82-based inbreds, contributes a very good yield to moisture ratioto the F₁ hybrids. This favorable yield to moisture ratio for itsmaturity and high level of production directly correlate to a higherincome per acre for the farmer as it relates to high yields with minimaldrying costs. In addition, the lower grain moisture observed at harvestcombined with competitive yield will allow the farmer to begin harvestearlier without sacrificing yield potential compared to later hybrids.F₁ hybrids with inbred corn line ML12 as one of the parent lines produceconsistent sized ears with generally 16 to 18 kernel rows. Soundagronomics, good stalks, good late season intactness in the F₁ hybrids,attractive hard-textured grain, and a very uniform look at harvest aresome of the observed qualities. The harvest appearance of a hybrid is avisual rating taken at the time of harvest. The harvest intactness andstalk quality allow farmers to harvest more efficiently and reduce fieldlosses compared to hybrids that have inferior stalks and poor planthealth.

Inbred corn line ML12 is similar to corn line LH185, however, there arenumerous differences. The inbred corn line ML12 consistency of eardevelopment, increased kernel row number, hard grain texture, and rapiddry down are transferred to the F₁ hybrids in the majority of crossesand are exhibited in a unique appearance, especially at harvest time.

Inbred corn line ML12 is a mid-season inbred. Heat units to 50% pollenshed are approximately 1594 and to 50% silk are also approximately 1594as measured near Ames, Iowa.

ML12 is an inbred corn line with high yield potential in hybrids.Hybrids with inbred corn line ML12 as one parental line produce uniform,consistent sized ears with a high kernel row number and heavy,hard-textured kernels. Often these hybrid combinations result in plantswhich are appreciably better than average for stalk strength, grainyield, and test weight when compared to inbred lines of similar maturityand geographical adaptability. Some of the criteria used to select earsin various generations include: yield, yield to harvest moisture ratio,stalk quality, root quality, disease tolerance with emphasis on greyleaf spot, test weight, late season plant greenness, late season plantintactness, ear retention, ear height, pollen shedding ability, Bilkingability, and corn borer tolerance. During the development and selectionof the line, crosses were made to inbred testers for the purpose ofestimating the line's general and specific combining ability, andevaluations were run by the Ames, Iowa Research Station. The inbred wasevaluated further as a line and in numerous crosses by the Ames stationand other research stations across the Corn Belt. The inbred has provento have an excellent combining ability in hybrid combinations.

Inbred corn line ML12 has shown uniformity and stability for the traits,within the limits of environmental influence for the traits. The linehas been increased with continued observation for uniformity of planttype. Inbred corn line ML12 has the following morphologic and othercharacteristics (based primarily on data collected at Ames, Iowa):

TABLE 1 VARIETY DESCRIPTION INFORMATION General Plant Information: 1.Type: ML12 is a yellow, dent corn inbred 2. Region where developed:Ames, Iowa 3. Maturity: Heat Units From planting to 50% of plants insilk: 1594 From planting to 50% of plants in pollen: 1594 Plant: 1.Plant height to tassel tip: 240.0 cm 2. Ear height to base of top ear:85 cm 3. Average length of top ear internode: 18.5 cm 4. Average numberof tillers: 0 5. Average number of ears per stalk: 2.0 6. Anthocyanin ofbrace roots: Absent Leaf: 1. Width of ear node leaf: 10.5 cm 2. Lengthof ear node leaf: 79 cm 3. Number of leaves above top ear: 6.0 4. Leafangle (from 2nd leaf above ear at anthesis to stalk above leaf): 27° 5.Leaf color: Munsell 10GY 4/4 6. Leaf sheath pubescence (Rated on scalefrom 1 = none to 9 = like peach fuzz): 1 7. Marginal waves (Rated onscale from 1 = none to 9 = many): 7 8. Longitudinal creases (Rated onscale from 1 = none to 9 = many): 1 Tassel: 1. Number of lateralbranches: 5.0 2. Branch angle from central spike: 22° 3. Tassel length(from top leaf collar to tassel top): 47.5 cm 4. Anther color: Munsell7.5Y 7/10 5. Glume color: Munsell 10Y 76/6 6. Tassel glume bands color:Absent Ear (Unhusked Data): 1. Silk color (3 days after emergence):Munsell 2.5 GY 8.5/10 2. Fresh husk color (25 days after 50% silking):Munsell 5GY 8/8 3. Dry husk color (65 days after 50% silking): Munsell2.5Y 9/2 4. Position of ear: Pendant 5. Husk tightness (Rated on scalefrom 1 = very loose to 9 = very tight): 4 6. Husk extension at harvest:29 mm Ear (Husked Ear Data): 1. Ear length: 20.9 cm 2. Ear diameter atmid-point: 4.5 cm 3. Ear weight: 189 g 4. Number of kernel rows: 14.5 5.Row alignment: Straight 6. Shank length: 13.4 cm 7. Ear taper: NoneKernel (Dried): 1. Kernel length: 11 mm 2. Kernel width: 9.5 mm 3.Kernel thickness: 5.7 mm 4. Hard endosperm color: Munsell 7.5YR 5/8 5.Endosperm type: Dent 6. Weight per 100 kernels (unsized sample): 39.9 gCob: 1. Cob diameter at mid-point: 26.5 mm 2. Cob color: Red, Munsell7.5R 4/8 3. Agronomic Traits: 1. Pre-anthesis brittle snapping: 0% 2.Pre-anthesis root lodging: 32.1%

FURTHER EMBODIMENTS OF THE INVENTION

This invention is also directed to methods for producing a corn plant bycrossing a first parent corn plant with a second parent corn plantwherein either the first or second parent corn plant is an inbred cornplant of inbred corn line ML12. Further, both first and second parentcorn plants can come from the inbred corn line ML12. Whenself-pollinated, or crossed with another inbred corn line ML12 plant,inbred corn line ML12 will be stable while when crossed with another,different corn line, an F₁ hybrid seed is produced. Such methods ofhybridization and self-pollination of corn are well known to thoseskilled in the art of corn breeding.

An inbred corn line has been produced through several cycles ofself-pollination and is therefore to be considered as a homozygous line.An inbred line can also be produced though the dihaploid system whichinvolves doubling the chromosomes from a haploid plant thus resulting inan inbred line that is genetically stable (homozygous) and can bereproduced without altering the inbred line. A hybrid variety isclassically created through the fertilization of an ovule from an inbredparental line by the pollen of another, different inbred parental line.Due to the homozygous state of the inbred line, the produced gametescarry a copy of each parental chromosome. As both the ovule and thepollen bring a copy of the arrangement and organization of the genespresent in the parental lines, the genome of each parental line ispresent in the resulting F₁ hybrid, theoretically in the arrangement andorganization created by the plant breeder in the original parental line.

As long as the homozygosity of the parental lines is maintained, theresulting hybrid cross is stable. The F₁ hybrid is then a combination ofphenotypic characteristics issued from two arrangement and organizationof genes, both created by one skilled in the art through the breedingprocess.

Still further, this invention also is directed to methods for producingan inbred corn line ML12-derived corn plant by crossing inbred corn lineML12 with a second corn plant and growing the progeny seed, andrepeating the crossing and growing steps with the inbred corn lineML12-derived plant from 0 to 7 times. Thus, any such methods using theinbred corn line ML12 are part of this invention: selfing, backcrosses,hybrid production, crosses to populations, and the like. All plantsproduced using inbred corn line ML12 as a parent are within the scope ofthis invention, including plants derived from inbred corn line ML12.Advantageously, the inbred corn line is used in crosses with other,different, corn inbreds to produce first generation (F₁) corn hybridseeds and plants with superior characteristics.

It should be understood that the inbred can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims. As used herein, the term plant includesplant cells, plant protoplasts, plant cell tissue cultures from whichcorn plants can be regenerated, plant calli, plant clumps and plantcells that are intact in plants or parts of plants, such as embryos,pollen, ovules, flowers, kernels, seeds, ears, cobs, leaves, husks,stalks, roots, root tips, anthers, silk and the like.

Duncan, et al., (Planta, 1985, 165:322-332) indicates that 97% of theplants cultured that produced callus were capable of plant regeneration.Subsequent experiments with both inbreds and hybrids produced 91%regenerable, callus that produced plants. In a further study in 1988,Songstad, et al. (Plant Cell Reports, 7:262-265) reports several mediaadditions that enhance regenerability of callus of two inbred lines.Other published reports also indicated that “nontraditional” tissues arecapable of producing somatic embryogenesis and plant regeneration. K. V.Rao et al., (Maize Genetics Cooperation Newsletter, 1986, 60:64-65)refer to somatic embryogenesis from glume callus cultures and B. V.Conger, et al. (Plant Cell Reports, 1987, 6:345-347) indicate somaticembryogenesis from the tissue cultures of corn leaf segments. Thus, itis clear from the literature that the state of the art is such thatthese methods of obtaining plants are routinely used and have a veryhigh rate of success.

Tissue culture of corn is also described in European Patent Application,publication 160,390 and in Green and Rhodes, Maize for BiologicalResearch, Plant Molecular Biology Association, Charlottesville, Va.,1982, 367-372. Thus, another aspect of this invention is to providecells which upon growth and differentiation produce corn plants havingthe physiological and morphological characteristics of inbred corn lineML12.

The utility of inbred corn line ML12 also extends to crosses with otherspecies. Commonly, suitable species will be of the family Graminaceae,and especially of the genera Zea, Tripsacum, Coix, Schlerachne,Polyloca, Chionachne, and Trilobachne, of the tribe Maydeae. Potentiallysuitable for crosses with ML12 may be the various varieties of grainsorghum, Sorghum bicolor (L.) Moench.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes.” Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed inbred line. An embodiment of the presentinvention comprises at least one transformation event in inbred cornline ML12.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid, and can beused alone or in combination with other plasmids, to provide transformedcorn plants, using transformation methods as described below toincorporate transgenes into the genetic material of the corn plant(s).

Expression Vectors for Corn Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which, when placed under the control of plant regulatory signals,confers resistance to kanamycin (Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983)). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin (Vanden Elzen et al., Plant Mol. Biol., 5:299(1985)).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant (Hayford et al., PlantPhysiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987),Svab et al., Plant Mol. Biol. 14:197 (1990), and Hille et al., PlantMol. Biol. 7:171 (1986)). Other selectable marker genes conferresistance to herbicides such as glyphosate, glufosinate or bromoxynil(Comai et al., Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell2:603-618 (1990) and Stalker et al., Science 242:419-423 (1988)).

Selectable marker genes for plant transformation that are not ofbacterial origin include, for example, mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase (Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shahet al., Science 233:478 (1986), and Charest et al., Plant Cell Rep.8:643 (1990)).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include beta-glucuronidase (GUS),beta-galactosidase, luciferase, and chloramphenicol acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al.,EMBO J. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci. U.S.A. 84:131(1987), and DeBlock et al., EMBO J. 3:1681 (1984). Another approach tothe identification of relatively rare transformation events has been useof a gene that encodes a dominant constitutive regulator of the Zea maysanthocyanin pigmentation pathway (Ludwig et al., Science 247:449(1990)).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are also available. However, these in vivomethods for visualizing GUS activity have not proven useful for recoveryof transformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

A gene encoding Green Fluorescent Protein (GFP) has been utilized as amarker for gene expression in prokaryotic and eukaryotic cells (Chalfieet al., Science 263:802 (1994)). GFP and mutants of GFP may be used asscreenable markers.

Expression Vectors for Corn Transformation: Promoters

Genes included in expression vectors must be driven by nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain organs,such as leaves, roots, seeds and tissues such as fibers, xylem vessels,tracheids, or sclerenchyma. Such promoters are referred to as“tissue-preferred.” Promoters which initiate transcription only incertain tissue are referred to as “tissue-specific.” A “cell-type”specific promoter primarily drives expression in certain cell types inone or more organs, for example, vascular cells in roots or leaves. An“inducible” promoter is a promoter which is under environmental control.Examples of environmental conditions that may effect transcription byinducible promoters include anaerobic conditions or the presence oflight. Tissue-specific, tissue-preferred, cell-type specific, andinducible promoters constitute the class of “non-constitutive”promoters. A “constitutive” promoter is a promoter which is active undermost environmental conditions.

A. Inducible Promoters—An inducible promoter is operably linked to agene for expression in corn. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in corn. With aninducible promoter the rate of transcription increases in response to aninducing agent. Any inducible promoter can be used in the instantinvention. See Ward et al., Plant Mol. Biol. 22:361-366 (1993).Exemplary inducible promoters include, but are not limited to, that fromthe ACEI system which responds to copper (Mett et al., Proc. Natl. Acad.Sci. U.S.A. 90:4567-4571 (1993)); In2 gene from maize which responds tobenzenesulfonamide herbicide safeners (Gatz et al., Mol. Gen. Genetics243:32-38 (1994)) or Tet repressor from Tn10 (Gatz et al., Mol. Gen.Genetics 227:229-237 (1991)). A particularly preferred induciblepromoter is a promoter that responds to an inducing agent to whichplants do not normally respond. An exemplary inducible promoter is theinducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone (Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991)).

B. Constitutive Promoters—A constitutive promoter is operably linked toa gene for expression in corn or the constitutive promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in corn. Many differentconstitutive promoters can be utilized in the instant invention.Exemplary constitutive promoters include, but are not limited to, thepromoters from plant viruses such as the 35S promoter from CaMV (Odellet al., Nature 313:810-812 (1985)) and the promoters from such genes asrice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin(Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensenet al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor.Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730(1984)) and maize H3 histone (Lepetit et al., Mol. Gen. Genetics231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3): 291-300(1992)).

The ALS promoter, XbaI/NcoI fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said XbaI/NcoIfragment), represents a particularly useful constitutive promoter. SeePCT application WO96/30530.

C. Tissue-specific or Tissue-preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in corn.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in corn. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zml3or a microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Knox, C., et al.,Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol. 91:124-129(1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell. Biol.108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon, etal., Cell 39:499-509 (1984), Stiefel, et al., Plant Cell 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is corn. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RFLP, PCR andSSR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defences are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant inbred line can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btalpha-endotoxin gene. Moreover, DNA molecules encoding alpha-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Numbers 40098, 67136, 31995 and31998.

C. A lectin. See, for example, the article by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT application US93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus alpha-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid orjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Pratt et al., Biochem. Biophys. Res. Comm. 163:1243(1989) (an allostatin is identified in Diploptera puntata). See alsoU.S. Pat. No. 5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific, paralytic neurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, a hydroxamic acid, a phenylpropanoidderivative or another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Numbers 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance).

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-beta, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruseS. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect.

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-alpha-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilising plantcell wall homo-alpha-1,4-D-galacturonase. See Lamb et al., BioTechnology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

R. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., BioTechnology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus (PAT bar genes), and pyridinoxy or phenoxy propionic acidsand cyclohexones (ACCase inhibitor-encoding genes). See, for example,U.S. Pat. No. 4,940,835 to Shah et al., which discloses the nucleotidesequence of a form of EPSP which can confer glyphosate resistance. A DNAmolecule encoding a mutant aroA gene can be obtained under ATCCaccession number 39256, and the nucleotide sequence of the mutant geneis disclosed in U.S. Pat. No. 4,769,061 to Comai. European patentapplication No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a PAT gene is provided in Europeanapplication No. 0 242 246 to Leemans et al. DeGreef et al.,BioTechnology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for PAT activity. Exemplary ofgenes conferring resistance to phenoxy propionic acids and cyclohexones,such as sethoxydim and haloxyfop are the ABC5-S1, ABC5-S2 and ABC5-S3genes described by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) or a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNumbers 53435, 67441, and 67442. Cloning and expression of DNA codingfor a glutathione S-transferase is described by Hayes et al., Biochem.J. 285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knutzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992)

B. Increased resistance to high light stress such as photo-oxidativedamages, for example by transforming a plant with a gene coding for aprotein of the Early Light Induced Protein family (ELIP) as described inWO 03/074713 in the name of Biogemma.

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bact. 170:810 (1988)(nucleotide sequence of Streptococcus mutants fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 20:220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al.,BioTechnology 10:292 (1992) (production of transgenic plants thatexpress Bacillus lichenifonnis α-amylase), Elliot et al., Plant Molec.Biol. 21:515 (1993) (nucleotide sequences of tomato invertase genes),Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley alpha-amylase gene), and Fisher et al., PlantPhysiol. 102:1045 (1993) (maize endosperm starch branching enzyme II).

D. Increased resistance/tolerance to water stress or drought, forexample, by transforming a plant to create a plant having a modifiedcontent in ABA-Water-Stress-Ripening-Induced proteins (ARS proteins) asdescribed in WO 01/83753 in the name of Biogemma, or by transforming aplant with a nucleotide sequence coding for a phosphoenolpyruvatecarboxylase as shown in WO 02/081714. The tolerance of corn to droughtcan also be increased by an overexpression of phosphoenolpyruvatecarboxylase (PEPC-C4), obtained, for example from sorghum.

E. Increased content of cysteine and glutathione, useful in theregulation of sulfur compounds and plant resistance against variousstresses such as drought, heat or cold, by transforming a plant with agene coding for an Adenosine 5′ Phosphosulfate as shown in WO 01/49855.

F. Increased nutritional quality, for example, by introducing a zeingene which genetic sequence has been modified so that its proteinsequence has an increase in lysine and proline. The increasednutritional quality can also be attained by introducing into the maizeplant an albumin 2S gene from sunflower that has been modified by theaddition of the KDEL peptide sequence to keep and accumulate the albuminprotein in the endoplasmic reticulum.

G. Decreased phytate content: 1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) A gene could be introduced thatreduced phytate content. In maize, this, for example, could beaccomplished, by cloning and then reintroducing DNA associated with thesingle allele which is responsible for maize mutants characterized bylow levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

4. Genes that Control Male Sterility

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT. See international publication WO 01/29237.

B. Introduction of various stamen-specific promoters. See internationalpublications WO 92/13956 and WO 92/13957.

C. Introduction of the barnase and the barstar genes. See Paul et al.,Plant Mol. Biol. 19:611-622, 1992).

Examples of Transgenes

MON810, also known as MON810Bt or BT1, is the designation given by theMonsanto Company (St. Louis, Mo.) for the transgenic event that, whenexpressed in maize, produces an endotoxin that is efficacious againstthe European corn borer, Ostrinia nubilalis and certain otherLepidopteran larvae.

MON603, also known as MON603RR2, better known as NK603, is thedesignation for the transgenic event that, when expressed in maize,allows the use of glyphosate as a weed control agent on the crop.Another transgenic event, GA21, when expressed in maize, also allows theuse of glyphosate as a weed control agent on the crop.

MON89034, a designation given by the Monsanto Company (St. Louis, Mo.)for the transgenic event that, when expressed in maize, produces anendotoxin that is efficacious against the European corn borer, Ostrinianubilalis, fall armyworm, Spodoptera frugiperda, and certain otherLepidopteran larvae.

MON88017, also known as MON88017CCR1, is the transgenic event that, whenexpressed in maize, allows the use of glyphosate as a weed controlagent. In addition, this event produces an endotoxin that is efficaciousagainst the corn root worm, Diabrotica virgifera, and certain otherColeopteran larvae.

HERCULEX Corn Borer, better known as HX1 or TC1507, is the designationfor the transgenic event that, when expressed in maize, produces anendotoxin that is efficacious against the European corn borer, Ostrinianubilalis, and certain other Lepidopteran larvae. In addition, thetransgenic event was developed to allow the crop to be tolerant to theuse of glufosinate ammonium, the active ingredient in phosphinothricinherbicides.

HERCULEX Root Worm, or DAS59122-7, is the designation for the transgenicevent that, when expressed in maize, produces an endotoxin that isefficacious against the corn root worm, Diabrotica virgifera, andcertain other Coleopteran larvae. In addition, the transgenic event wasdeveloped to allow the crop to be tolerant to the use of glufosinateammonium, the active ingredient in phosphinothricin herbicides.

T25 is the designation for the transgenic event that, when expressed inmaize, allows the crop to be tolerant to the use of glufosinateammonium, the active ingredient in phosphinothricin herbicides.

Methods for Corn Transformation

Numerous Methods For Plant Transformation Have Been Developed, IncludingBiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch et al., Science227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra, andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer—Despite the fact the host range forAgrobacterium-mediated transformation is broad, some major cereal cropspecies and gymnosperms have generally been recalcitrant to this mode ofgene transfer, even though some success has recently been achieved inrice and corn. Hiei et al., The Plant Journal 6:271-282 (1994) and U.S.Pat. No. 5,591,616 issued Jan. 7, 1997. Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 micron. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299(1988), Klein et al., BioTechnology 6:559-563 (1988), Sanford, J. C.,Physiol Plant 7:206 (1990), Klein et al., BioTechnology 10:268 (1992).In corn, several target tissues can be bombarded with DNA-coatedmicroprojectiles in order to produce transgenic plants, including, forexample, callus (Type I or Type II), immature embryos, and meristematictissue.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., BioTechnology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. D'Halluin et al., Plant Cell 4:1495-1505 (1992) andSpencer et al., Plant Mol. Biol. 24:51-61 (1994).

Following transformation of corn target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic inbred line. The transgenic inbred line couldthen be crossed, with another (non-transformed or transformed) inbredline, in order to produce a new transgenic inbred line. Alternatively, agenetic trait which has been engineered into a particular corn lineusing the foregoing transformation techniques could be moved intoanother line using traditional backcrossing techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachcould be used to move an engineered trait from a public, non-eliteinbred line into an elite inbred line, or from an inbred line containinga foreign gene in its genome into an inbred line or lines which do notcontain that gene. As used herein, “crossing” can refer to a simple X byY cross, or the process of backcrossing, depending on the context.

When the term inbred corn plant is used in the context of the presentinvention, this also includes any inbred corn plant where one or moredesired traits have been introduced through backcrossing methods,whether such trait is a naturally occurring one or a transgenic one.Backcrossing methods can be used with the present invention to improveor introduce one or more characteristic into the inbred. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to one of the parental corn plants for that inbred. Theparental corn plant which contributes the gene or the genes for thedesired characteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental corn plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Fehr, 1987).

In a typical backcross protocol, the original inbred of interest(recurrent parent) is crossed to a second inbred (nonrecurrent parent)that carries the gene or genes of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a corn plant isobtained wherein all the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant in addition to the gene or genes transferred from the nonrecurrentparent. It should be noted that some, one, two, three or more,self-pollination and growing of a population might be included betweentwo successive backcrosses. Indeed, an appropriate selection in thepopulation produced by the self-pollination, i.e., selection for thedesired trait and physiological and morphological characteristics of therecurrent parent might be equivalent to one, two or even threeadditional backcrosses in a continuous series without rigorousselection, saving time, money and effort for the breeder. The backcrossprocess could also be accelerated through a step of haploid inductiontogether by a molecular marker screening to identify the backcrossprogeny plants that have the closest genetic resemblance with therecurrent line, together with the gene or genes of interest to betransferred. A non limiting example of such a protocol would be thefollowing: a) the first generation F₁ produced by the cross of therecurrent parent A by the donor parent B is backcrossed to parent A, b)selection is practiced for the plants having the desired trait of parentB, c) selected plants are self-pollinated to produce a population ofplants where selection is practiced for the plants having the desiredtrait of parent B and the physiological and morphologicalcharacteristics of parent A, d) the selected plants are backcrossed one,two, three, four, five or more times to parent A to produce selectedbackcross progeny plants comprising the desired trait of parent B andthe physiological and morphological characteristics of parent A. Step c)may or may not be repeated and included between the backcrosses of stepd. Step c) may or may not be followed by a step of haploid inductionfollowed by molecular marker screening.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute one or more trait(s) or characteristic(s) in theoriginal inbred. To accomplish this, a gene or genes of the recurrentinbred is modified or substituted with the desired gene or genes fromthe nonrecurrent parent, while retaining essentially all of the rest ofthe desired genetic, and therefore the desired physiological andmorphological, constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait(s) to the plant. The exactbackcrossing protocol will depend on the characteristic(s) or trait(s)being altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a single gene and dominant allele, multiple genes andrecessive allele(s) may also be transferred and therefore, backcrossbreeding is by no means restricted to character(s) governed by one or afew genes. In fact the number of genes might be less important than theidentification of the character(s) in the segregating population. Inthis instance it may then be necessary to introduce a test of theprogeny to determine if the desired characteristic(s) has beensuccessfully transferred. Such tests encompass not only visualinspection and simple crossing, but also follow up of thecharacteristic(s) through genetically associated markers and molecularassisted breeding tools. For example, selection of progeny containingthe transferred trait is done by direct selection, visual inspection fora trait associated with a dominant allele, while the selection ofprogeny for a trait that is transferred via a recessive allele, such asthe waxy starch characteristic, require selling the progeny to determinewhich plant carry the recessive allele(s).

Many single gene traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic, i.e., they may be naturally present in the nonrecurrentparent, examples of these traits include but are not limited to, malesterility, waxy starch, amylose starch, herbicide resistance, resistancefor bacterial, fungal, or viral disease, insect resistance, malefertility, water stress tolerance, enhanced nutritional quality,industrial usage, increased digestibility yield stability and yieldenhancement. An example of this is the Rp1D gene which controlsresistance to rust fungus by preventing P. sorghi from producing spores.The Rp1D gene is usually preferred over the other Rp genes because it iswidely effective against all races of rust, but the emergence of newraces has lead to the use of other Rp genes comprising, for example, theRp1E, Rp1G, Rp1I, Rp1K or “compound” genes which combine two or more Rpgenes including Rp1GI, Rp1GDJ, etc. These genes are generally inheritedthrough the nucleus. Some known exceptions to this are the genes formale sterility, some of which are inherited cytoplasmically, but stillact as single gene traits. Several of these single gene traits aredescribed in U.S. Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, thedisclosures of which are specifically hereby incorporated by reference.

In 1981, the backcross method of breeding accounted for 17% of the totalbreeding effort for inbred line development in the United States,according to, Hallauer, A. R. et al., (1988) “Corn Breeding” in Corn andCorn Improvement, No. 18, pp. 463-481. The backcross breeding methodprovides a precise way of improving varieties that excel in a largenumber of attributes but are deficient in a few characteristics (Allard,1960, Principles of Plant Breeding, John Wiley & Sons, Inc.). The methodmakes use of a series of backcrosses to the variety to be improvedduring which the character or the characters in which improvement issought is maintained by selection. At the end of the backcrossing, thegene or genes being transferred unlike all other genes will beheterozygous. Selfing after the last backcross produces homozygosity forthis gene pair(s) and, coupled with selection, will result in a varietywith exactly the adaptation, yielding ability and qualitycharacteristics of the recurrent parent but superior to that parent inthe particular characteristic(s) for which the improvement program wasundertaken. Therefore, this method provides the plant breeder with ahigh degree of genetic control of his work.

Backcrossing is a powerful mechanism for achieving homozygosity and anypopulation obtained by backcrossing may rapidly converge on the genotypeof the recurrent parent. When backcrossing is made the basis of a plantbreeding program, the genotype of the recurrent parent will be modifiedwith regards to genes being transferred, which are maintained in thepopulation by selection.

Examples of successful backcrosses are the transfer of stem rustresistance from “Hope” wheat to “Bart” wheat and the transfer of buntresistance to “Bart” wheat to create “Bart 38”which has resistance toboth steprn rust and bunt. Also highlighted by Allard is the successfultransfer of mildew, leaf spot and wilt resistances in “CaliforniaCommon” alfalfa to create “Caliverde.” This new “Caliverde” varietyproduced through the backcross process is indistinguishable from“California Common” except for its resistance to the three nameddiseases.

One of the advantages of the backcross method is that the breedingprogram can be carried out in almost every environment that will allowthe development of the character being transferred. Another advantage ofthe backcross method is that more than one character or trait can betransferred, either through several backcrosses or through the use oftransformation and then backcrossing.

The backcross technique is not only desirable when breeding for diseaseresistance but also for the adjustment of morphological characters,color characteristics and simply inherited quantitative characters suchas earliness, plant height and seed size and shape. In this regard, amedium grain type variety, “Calady,” has been produced by Jones andDavis. In dealing with quantitative characteristics, they selected thedonor parent with the view of sacrificing some of the intensity of thecharacter for which it was chosen, i.e., grain size. “Lady Wright,” along grain variety was used as the donor parent and “Coloro,” a shortgrain one as the recurrent parent. After four backcrosses, the mediumgrain type variety “Calady” was produced.

DEPOSIT INFORMATION

A deposit of the inbred corn line of this invention is maintained byAgReliant Genetics, LLC, 4640 East SR32, Lebanon, Indiana 46052.AgReliant maintains the seed deposit on behalf of Limagrain Europe andKWS SAAT AG. In addition, a sample of the inbred corn seed of thisinvention has been or will be deposited with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110 or theNational Collections of Industrial, Food and Marine Bacteria (NCIMB), 23St Machar Drive, Aberdeen, Scotland, AB24 3Ry, United Kingdom.

To satisfy the enablement requirements of 35 U.S.C. 112, and to certifythat the deposit of the isolated strain (i.e., corn inbred) of thepresent invention meets the criteria set forth in 37 CFR 1.801-1.809,Applicants hereby make the following statements regarding the depositedcorn inbred line ML12 (deposited as ATCC Accession No. PTA-120628 onOct. 16, 2013):

1. During the pendency of this application, access to the invention willbe afforded to the Commissioner upon request;

2. All restrictions on availability to the public will be irrevocablyremoved upon granting of the patent under conditions specified in 37 CFR1.808;

3. The deposit will be maintained in a public repository for a period of30 years or 5 years after the last request or for the effective life ofthe patent, whichever is longer;

4. A test of the viability of the biological material at the time ofdeposit will be conducted by the public depository under 37 CFR 1.807;and

5. The deposit will be replaced if it should ever become unavailable.

Access to this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. Upon granting of any claims in this application, all restrictionson the availability to the public of the variety will be irrevocablyremoved by affording access to a deposit of at least 2,500 seeds of thesame variety with the ATCC or NCIMB.

INDUSTRIAL APPLICABILITY

Corn is used as human food, livestock feed, and as raw material inindustry. The food uses of corn, in addition to human consumption ofcorn kernels, include both products of dry- and wet-milling industries.The principal products of corn dry-milling are grits, meal and flour.Corn meal is flour ground to fine, medium, and coarse consistencies fromdried corn. In the United States, the finely ground corn meal is alsoreferred to as corn flour. However, the term “corn flour” denotes cornstarch in the United Kingdom. Corn meal has a long shelf life and isused to produce an assortment of products, including but not limited totortillas, taco shells, bread, cereal and muffins.

The corn wet-milling industry can provide corn starch, corn syrups, cornsweeteners and dextrose for food use. Corn syrup is used in foods tosoften texture, add volume, prevent crystallization of sugar and enhanceflavor. Corn syrup is distinct from high-fructose corn syrup (HFCS),which is created when corn syrup undergoes enzymatic processing,producing a sweeter compound that contains higher levels of fructose.

Corn oil is recovered from corn germ, which is a by-product of both dry-and wet-milling industries. Corn oil which is high in mono and polyunsaturated fats, is used for reducing fat and trans fat in numerousfood products.

Corn, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs and poultry.

Industrial uses of corn include production of ethanol, corn starch inthe wet-milling industry and corn flour in the dry-milling industry.Corn ethanol is ethanol produced from corn as a biomass throughindustrial fermentation, chemical processing and distillation. Corn isthe main feedstock used for producing ethanol fuel in the United States.The industrial applications of corn starch and flour are based onfunctional properties, such as viscosity, film formation, adhesiveproperties, and ability to suspend particles. Corn starch and flour alsohave application in the paper and textile industries. Other industrialuses include applications in adhesives, building materials, foundrybinders, laundry starches, explosives, oil-well muds and other miningapplications.

Plant parts other than the grain of corn are also used in industry, forexample, stalks and husks are made into paper and wallboard and cobs areused for fuel and to make charcoal.

The seed of inbred corn line ML 12, the plant produced from the inbredseed, the hybrid corn plant produced from the crossing of the inbred,hybrid seed, and various parts of the hybrid corn plant and transgenicversions of the foregoing, can be utilized for human food, livestockfeed, and as a raw material in industry.

Tables of Field Test Trials

In the tables that follow, exemplary traits and characteristics ofhybrid combinations having inbred corn line ML12 as a parental line aregiven compared to other hybrids. The data collected are presented forkey characteristics and traits. The field tests are experimental trialsand have been made at numerous locations, with one or two replicationsper location under supervision of the applicant. Information about theseexperimental hybrids as compared to the check hybrids is presented.

There may be a pedigree listed in the comparison group of the hybrid(s)containing an inbred parent with the nomenclatures MON88017, BT1, RR2,CRW2, etc. following the inbred name. Such designations of thetransgenes are mentioned herein above. Information for each pedigreeincludes:

-   -   1. Mean yield of the hybrid across all locations (bushels/acre)        is shown in column 2.    -   2. A mean for the percentage moisture (H2O Grain) for the hybrid        across all locations is shown in column 3.    -   3. A mean of the yield divided by the percentage moisture (Y/M)        for the hybrid across all locations is shown in column 4.    -   4. A mean of the percentage of plants with stalk lodging (SL %)        across all locations is shown in column 5.    -   5. A mean of the percentage of plants with root lodging (RL %)        across all locations is shown in column 6.    -   6. Test weight (TW) is the grain density as measured in pounds        per bushel and is shown in column 7.    -   7. Harvest Appearance (Asp) is a rating made by a trained person        on the date of harvest. Harvest appearance is the rater's        impression of the hybrid based on, but not limited to, a        combination of factors to include plant intactness, tissue        health appearance and ease of harvest as it relates to stalk        lodging and root lodging. A scale of 1=Lowest to 9=Highest/Most        Desirable is used and is listed in column 8.    -   8. Plant Height (PlHt) is a physical measurement taken from the        ground level to the tip of the tassel. It is expressed to the        nearest tenth of an inch and is shown in column 9.    -   9. Ear Height (EHt) is a physical measurement taken from the        ground level to the node of attachment for the upper ear. It is        expressed to the nearest tenth of an inch and is shown in column        10.

TABLE 2 Overall Comparisons: First year field trials, 4 locations HybridYLD H2O Y/M SL % RL % TW Asp PlHt EHt RBO1/LH287BT1 197.0 19.6 10.1 0.01.0 56.3 5.0 110.2 37.4 RBO1/LH185 191.2 18.4 10.4 1.6 0.4 57.3 4.7108.3 37.4 (HC33 & RBO1) & ML12 182.0 19.5 9.3 0.7 3.3 56.4 5.2 114.245.3 Pioneer 34H31 180.2 20.0 9.0 1.6 −0.9 56.7 5.0 110.2 43.3 (HC33 &RBO1) & UNW1 176.1 19.4 9.1 1.2 −0.9 57.2 5.0 106.3 47.2 Pioneer 34M94166.8 19.0 8.8 0.3 7.8 57.3 4.5 118.1 49.2 LH244/LH295 159.6 18.3 8.72.0 0.6 57.9 4.0 106.3 45.3 HC33/LH283 166.9 21.9 7.6 1.7 3.9 55.4 5.8112.2 45.3

TABLE 3 Overall Comparisons: Second year field trials, 8 locationsPedigree YLD H2O Y/M SL % RL % TW Asp PlHt EHt RBO1/ML12 192.6 19.2 10.00.7 0.1 56.0 5.5 109.2 41.3 BB14/ML12 193.3 19.6 9.9 1.6 3.5 55.9 5.8107.2 42.3 BB14/LH287BT1 185.9 20.5 9.1 2.8 1.9 55.1 5.5 104.2 36.4Pioneer 35Y64 178.8 18.8 9.5 0.3 2.1 56.7 5.2 109.2 45.2 RBO1/LH287BT1181.1 20.7 8.7 0.6 0.0 55.5 5.6 105.4 34.2 LH244/LH295 165.3 18.6 8.91.6 9.9 56.7 4.8 107.2 41.3 RBO1/LH185 155.7 18.8 8.3 0.7 0.6 56.2 4.993.4 34.4

TABLE 4 Overall Comparisons: Third year field trials, 9 locations HybridYLD H2O Y/M SL % RL % TW Asp PlHt EHt DeKalb DKC63-39 209.5 19.6 10.70.3 0.0 56.4 6.1 102.4 44.3 BB14/LH287BT1CRW2 203.0 17.9 11.3 1.0 0.056.9 5.9 103.3 39.4 RBO1/ML12 198.4 17.0 11.7 1.7 0.0 57.4 6.3 102.438.4 HCL205RR2/HCL607BT 191.5 15.1 12.7 2.2 0.4 58.4 5.4 107.3 46.3SGI890HX1/LH287 200.4 20.3 9.8 1.4 4.3 55.7 6.4 110.2 48.2 BB14/ML12193.2 17.7 10.9 1.8 0.0 57.0 6.1 103.3 44.3 Pioneer 34A16 193.0 18.210.6 2.4 2.0 57.1 6.1 108.3 44.3

TABLE 5 Overall Comparisons: Fourth year field trials, 8 locationsHybrid YLD H2O Y/M SL % RL % TW Asp PlHt EHt BB38/ML12 211.6 19.6 10.82.7 1.4 55.7 5.7 122.0 54.1 BB38/LH287BT1CCR1 211.0 20.5 10.3 1.3 0.255.1 4.8 118.1 48.2 BB14/LH287BT1CCR1 202.5 18.8 10.8 1.4 0.9 56.7 3.5109.3 44.3 BC5/LH287BT1CCR1 194.9 20.2 9.6 6.6 10.7 54.7 4.7 111.2 47.2CB1/UNW1BT1 194.2 19.9 9.8 6.8 10.4 55.8 5.0 114.2 51.2 CB1/MN5 184.120.5 9.0 2.8 0.2 54.8 4.7 119.1 51.2

TABLE 6 Overall Comparisons: Fifth year field trials, 19 locationsHybrid YLD H2O Y/M SL % RL % TW Asp PlHt EHt BC110/ML12 207.2 24.5 8.44.8 4.6 52.0 5.1 124.0 50.9 BB46/MM27BT1 199.0 26.7 7.4 2.3 4.1 51.3 6.2116.8 48.9 BB38/LH287RR2 196.3 27.4 7.2 0.6 0.2 50.9 6.0 116.5 42.7HCL301CCR1/HCL516BT1 192.8 24.6 7.8 1.2 4.3 52.1 5.2 115.2 45.3BB14RR2/MM27 189.9 25.4 7.5 2.4 0.3 51.1 5.0 112.5 39.4 BB14CCR1/MM27BT1189.1 26.8 7.0 1.3 0.0 51.1 6.1 116.8 46.3 HCL301RR2/HCL516 188.2 25.07.5 0.7 1.1 51.9 5.4 117.5 44.3

TABLE 7 Overall Comparisons: Fifth year field trials, 23 locationsPedigree YLD H2O Y/M SL % RL % TW Asp PlHt EHt BC115/ML12 220.0 23.8 9.31.4 8.3 52.8 5.7 124.5 54.3 DeKalb DKC61-69 210.1 25.6 8.2 2.3 3.9 52.65.4 115.0 50.2 RBO1RR/LH287BT 198.7 26.0 7.7 0.8 0.7 52.8 5.7 115.4 40.8BB14BTRR/MN7 198.7 24.9 8.0 0.8 0.4 52.1 5.3 114.6 46.3 BB14RR/MM27197.8 23.9 8.3 1.8 0.1 53.0 5.5 113.4 40.8 HCL301RR/HCL516 193.2 24.57.9 1.7 0.9 52.6 5.4 118.6 48.7

TABLE 8 Overall Comparisons: Sixth year field trials, 26 locationsHybrid YLD H2O Y/M SL % RL % TW Asp PlHt EHt BB59/LH287RR2 187.8 19.19.8 1.6 0.3 55.3 6.2 111.7 38.5 BB36/ML12 191.1 20.5 9.3 3.6 4.1 55.06.2 121.1 44.7 BB14/MM27 181.0 18.6 9.7 2.7 0.9 55.4 5.6 110.9 37.6DeKalb DKC61-69 183.2 19.5 9.4 3.3 2.3 55.4 5.8 113.4 44.4 BB38/ML9177.8 18.7 9.5 1.1 0.3 55.4 6.3 113.0 37.6 RBO1RR2/LH287 177.8 19.3 9.22.1 2.5 55.5 5.5 109.2 37.4 HCL301RR2/HCL516BT1 171.5 17.8 9.6 3.7 2.755.9 5.6 114.2 40.0

TABLE 9 Overall Comparisons: Sixth year field trials, 28 locationsHybrid YLD H2O Y/M SL % RL % TW Asp PlHt EHt RBO1BT1CCR1/ML9 186.5 17.011.0 2.1 1.2 56.2 6.3 99.9 36.2 BC110/ML12 189.1 19.5 9.7 3.0 4.1 55.65.7 103.9 41.4 DeKalb DKC52-59 178.4 16.4 10.9 3.3 1.4 56.2 5.9 93.640.1 HCL301RR2/HCL516BT1 183.3 18.8 9.7 3.2 2.1 55.8 5.8 99.2 38.1Pioneer 35F44 182.3 19.5 9.3 0.5 1.0 56.3 6.3 95.1 36.3 R6258RMQKZ ×R2683LMSLZ 175.6 19.0 9.2 5.2 2.7 55.8 6.0 103.6 43.1

TABLE 10 Overall Comparisons: Seventh year field trials, 17 locationsHybrid YLD H2O Y/M SL % RL % TW Asp PlHt EHt F0297RMQKZ/ML12 188.2 20.49.2 0.5 3.8 57.1 5.7 115.8 44.9 BB38/ML9 182.7 19.8 9.2 0.3 0.3 56.5 5.7105.8 38.8 BB59/LH287BT1CCR1 186.5 21.6 8.7 0.0 3.5 55.9 6.1 107.4 38.6Pioneer 0916XR 187.1 20.4 9.2 0.1 7.9 58.1 5.8 103.4 39.8 DeKalbDKC61-69 177.6 21.2 8.4 0.1 10.5 56.9 5.4 101.9 38.9 DeKalb DKC57-50179.2 20.0 8.9 1.1 −0.1 57.1 5.5 108.2 42.1 BB59/LH287BT1CCR1 183.2 21.78.4 0.6 3.9 55.8 6.2 105.9 38.5

The foregoing detailed description has been given for clearness ofunderstanding only and no unnecessary limitations should be understoodthere from as modifications will be obvious to those skilled in the art.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materials,similar or equivalent to those described herein, can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications, patents, and patentpublications cited are incorporated by reference herein in theirentirety for all purposes.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

What is claimed is:
 1. A seed of inbred corn line designated ML12,wherein a representative sample of seed of said line was deposited underATCC Accession No. PTA-120628.
 2. A corn plant, or a part thereof,produced by growing the seed of claim
 1. 3. A corn plant, or a partthereof, having all the physiological and morphological characteristicsof inbred line ML12, wherein a representative sample of seed of saidline was deposited under ATCC Accession No. PTA-120628.
 4. A tissueculture of cells produced from the plant of claim
 2. 5. A corn plantregenerated from the tissue culture of claim 4, wherein the regeneratedplant has all the morphological and physiological characteristics ofinbred line ML12, wherein a representative sample of seed of said linewas deposited under ATCC Accession No. PTA-120628.
 6. A method forproducing a hybrid corn seed wherein the method comprises crossing theplant of claim 2 with a different corn plant and harvesting theresultant hybrid corn seed.
 7. A hybrid corn seed produced by the methodof claim
 6. 8. A hybrid corn plant, or part thereof, produced by growingthe seed of claim
 7. 9. A method for producing inbred corn line ML12,wherein a representative sample of seed of said line was deposited underATCC Accession No. PTA-E120628, wherein the method comprises: a)planting a collection of seeds comprising seed of a hybrid, one of whoseparents is inbred line ML12, said collection also comprising seed ofsaid inbred; b) growing plants from said collection of seeds; c)identifying the plants having the physiological and morphologicalcharacteristics of inbred corn line ML12 as inbred parent plants; d)controlling pollination of said inbred parent plants in a manner whichpreserves the homozygosity of said inbred parent plant; and e)harvesting the resultant seed.
 10. The method of claim 9 wherein step(c) comprises identifying plants with decreased vigor compared to theother plants grown from the collection of seeds.
 11. A method forproducing a corn plant that contains in its genetic material one or moretransgenes, wherein the method comprises crossing the corn plant ofclaim 2 with either a second plant of another corn line which contains atransgene or a transformed corn plant of the inbred corn line ML12, sothat the genetic material of the progeny that results from the crosscontains the transgene(s) operably linked to a regulatory element andwherein the transgene is selected from the group consisting of malesterility, male fertility, herbicide resistance, insect resistance,disease resistance, water stress tolerance, and increased digestibility.12. A corn plant, or a part thereof, produced by the method of claim 11.13. The corn plant of claim 12, wherein the transgene confers resistanceto an herbicide selected from the group consisting of imidazolinone,sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine andbenzonitrile.
 14. The corn plant of claim 12, wherein the transgeneencodes a Bacillus thuringiensis protein.
 15. The corn plant of claim12, wherein the transgene confers disease resistance.
 16. The corn plantof claim 12, wherein the transgene confers water stress tolerance. 17.The corn plant of claim 12, where in the transgene confers increaseddigestibility.
 18. A method for producing a hybrid corn seed wherein themethod comprises crossing the plant of claim 12 with a different cornplant and harvesting the resultant hybrid corn seed.
 19. A method ofproducing a corn plant with waxy starch or increased amylose starchwherein the method comprises transforming the corn plant of claim 2 witha transgene that modifies carbohydrate metabolism.
 20. A corn plantproduced by the method of claim
 19. 21. A method of introducing one ormore desired traits into inbred corn line ML12 wherein the methodcomprises: a) crossing the inbred line ML12 plants grown from the inbredline ML12 seed, wherein a representative sample of seed of said line wasdeposited under ATCC Accession No. PTA-120628, with plants of anothercorn line that comprise one or more desired traits to produce progenyplants, wherein the one or more desired traits are selected from thegroup consisting of male sterility, male fertility, herbicideresistance, insect resistance, disease resistance, waxy starch, waterstress tolerance, increased amylose starch and increased digestibility;b) selecting progeny plants that have the one or more desired traits toproduce selected progeny plants; c) crossing the selected progeny plantswith the inbred corn line ML12 plants to produce backcross progenyplants; d) selecting for backcross progeny plants that have the one ormore desired traits and physiological and morphological characteristicsof inbred corn line ML12 listed in Table 1 to produce selected backcrossprogeny plants; and e) repeating steps (c) and (d) one or more times insuccession to produce selected second or higher backcross progeny plantsthat comprise the desired one or more traits and the physiological andmorphological characteristics of inbred corn line ML12 as listed inTable
 1. 22. A corn plant produced by the method of claim 21, whereinthe plant has the one or more desired traits and all of thephysiological and morphological characteristics of inbred corn line ML12as listed in Table
 1. 23. A method for producing inbred corn line ML12seed, wherein a representative sample of seed of said line was depositedunder ATCC Accession No. PTA-120628, wherein the method comprisescrossing a first inbred parent corn plant with a second inbred parentcorn plant and harvesting the resultant corn seed, wherein both saidfirst and second inbred corn plant are the corn plant of claim
 2. 24. Amethod for producing inbred corn line ML12 seed, wherein arepresentative sample of seed of said line was deposited under ATCCAccession No. PTA-120628, wherein the method comprises: a) planting theinbred corn seed of claim 1; b) growing a plant from said seed; c)controlling pollination in a manner that the pollen produced by thegrown plant pollinates the ovules produced by the grown plant; and d)harvesting the resultant seed.
 25. A method for producing a corn seedthat contains in its genetic material one or more transgenes, whereinthe method comprises crossing the corn plant of claim 2 with either asecond plant of another corn line which contains one or more transgenesor a transformed corn plant of the inbred corn line ML12, wherein thetransgene(s) is operably linked to a regulatory element and wherein thetransgene is selected from the group consisting of male sterility, malefertility, herbicide resistance, insect resistance, disease resistance,water stress tolerance, and increased digestibility; and harvesting theresultant seed.
 26. A corn seed produced by the method of claim 25, or acell of said seed.
 27. A method for producing a hybrid corn seed whereinthe method comprises crossing the plant of claim 22 with a differentcorn plant and harvesting the resultant hybrid corn seed.
 28. A hybridcorn seed produced by the method of claim
 27. 29. A corn plant producedby the method of claim 21, wherein the plant has the one or more desiredtraits and all of the physiological and morphological characteristics ofinbred corn line ML12 as listed in Table 1, except said one or moredesired traits.